DESCRIPTION (from applicant's abstract) GABA is the major inhibitory neurotransmitter in the brain. Fast inhibitory post-synaptic potentials are mediated by GABAA receptors (GABARs), which contain binding sites for many clinically relevant drugs such as benzodiazepines, barbiturates, and general anesthetics. GABAR currents are also modulated by neurosteroids and lanthanum and antagonized by penicillin, picrotoxin, bicuculline, furosemide, and zinc. The GABAR is a hetero-oligomeric protein complex composed of five subunits which together form a transmembrane chloride ion channel. Four different subunit families (alpha, beta, gamma, delta) have been studied extensively and two new subunit families pi and epsilon have been identified recently. Each subunit family is composed of one or more subtypes. Six alpha (alpha1-alpha6), three beta (beta1-beta3), three gamma(gamma1-gamma3), and delta(delta1), one e (e1) and one pi(pi1) subunit subtypes have been identified. Pharmacological studies of recombinant receptors have shown that individual subtypes confer different sensitivities to GABAR modulators such as benzodiazepines, barbiturates, propofol, loreclezole, alcohol, furosemide, zinc, other divalent cations and lanthanum. The hypotheses to be tested are the following: 1) GABAR subunit subtypes contain binding and modulatory sites that are subtype specific. 2) Allosteric modulators bind to N-terminal, extracellular portions of M2 or M2-M2 extracellular domains. 3) Binding of allosteric modulators bind to a restricted number of amino acid residues on these extracellular domains. 4) The kinetic properties of GABARs, including gating and desensitization, are subunit subtype specific. 5) Specific functional domains are present in the transmembrane portion of GABAR subunit subtypes that determine their kinetic properties. The specific aims are to determine: 1) Binding site(s) on GABAR beta and/or alpha subtypes for zinc and other divalent cations. 2) Modulatory sites on alpha, gamma, delta, and epsilon subtypes that regulate sensitivity to zinc and other divalent cations. 3) Binding sites on GABAR subtypes for lanthanum enhancement and inhibitions of GABAR current. 4) Binding sites on GABAR subtypes for furosemide inhibition of GABAR current. 5) Binding sites on GABAR subtypes for barbiturate enhancement of GABAR current and direct activation of current. 6) Biophysical properties of recombinant GABAR isoforms assembled from GABAR subtypes that are expressed in hippocampal dentate granule cells. 7) Structural bases for the biophysical properties of recombinant GABAR isoforms that are assembled from GABAR subtypes expressed in hippocampal dentate granule cells.